Characterization of Polysaccharides from the Pericarp of Zanthoxylum bungeanum Maxim by Saccharide Mapping and Their Neuroprotective Effects

The pericarp of Zanthoxylum bungeanum maxim (PZM) is a commonly used spice and herbal medicine in China. In the present study, the structural characteristics of PPZM were investigated by saccharide mapping after enzymatic digestion by using high-performance thin layer chromatography (HPTLC) and polysaccharide analysis by using carbohydrate gel electrophoresis (PACE). The mechanisms of protective effects of PPZM on Aβ25–35-induced oxidative damage were explored in PC12 cells. The results showed that PPZM contained 1,4-α-D-galactosidic, 1,4-α-D-galactosiduronic, and (1→4)-β-D-glucosidic linkages. Pretreatment with PPZM significantly increased the cell viability of Aβ25–35-injured PC12 cells. Flow cytometry and Hoechst/PI staining indicated that PPZM gradually relieved the apoptosis of the Aβ25–25-treated cells. PPZM markedly decreased the ROS level of PC12 cells and suppressed Aβ25–35-induced oxidative stress by increasing the SOD level, and decreasing the level of MDA and LDH. The mRNA expressions of caspase-3 and Bax were significantly downregulated, and Bcl-2 expression was upregulated by treatment with PPZM. PPZM significantly increased the mRNA expression of Nrf2 and HO-1 in Aβ25–35 treated cells. The results indicated that PPZM alleviated apoptosis and oxidative stress induced by Aβ25–25 through the inhibition of mitochondrial dependent apoptosis and activation of Nrf2/HO-1 pathway. PPZM can be used as a potential protective agent against Aβ25–25-induced neurotoxicity.


Introduction
Neurodegenerative diseases caused by aging seriously affect human health and quality of life in modern society [1]. Alzheimer's disease (AD) is a chronic neurodegenerative disease closely related to memory and cognitive impairment [2]. The pathological mechanism of AD is still unclear, and there is no satisfactory treatment plan at present [3]. The main pathological features related to AD include β-amyloid (Aβ) plaque, neurogenic fiber tangle and neuron loss [4]. Several hypotheses have been proposed to explain the causes of AD, including the cholinergic hypothesis, Aβ hypothesis and Tau protein hypothesis [5]. Despite continuous debate about the Aβ hypothesis, evidence supports Aβ plays a significant role in the pathogenesis of AD [6,7]. In addition, oxidative stress is an early event in the progression from normal aging to AD pathology, and is considered to be a key harmful factor of AD. Aβ and oxidative stress are linked to each other, because Aβ induces oxidative stress, and oxidative stress increases the Aβ deposition. Studies have shown that the gradual accumulation of oxidative damage for a long time will lead to the appearance of clinical and pathological AD symptoms, including Aβ deposition [8,9].
Excessive reactive oxygen species (ROS) can lead to cell apoptosis, and many fatal diseases are related to abnormal ROS. The brain is the most vulnerable to oxygen free

PPZM Protected PC12 Cells from Aβ25-35 Induced Cytotoxicity
As shown in Figure 2A, cells showed good cellular growth morphology in the control group without any treatment. However, after the incubation of Aβ25-25, the cell morphology changed, including the reduced cell quantity, membrane blebbing and cell shrinkage [22]. When pretreated with PPZM for 24 h, the morphology of the cells was observably improved, the above cell injuries alleviated.

PPZM Protected PC12 Cells from Aβ 25-35 Induced Cytotoxicity
As shown in Figure 2A, cells showed good cellular growth morphology in the control group without any treatment. However, after the incubation of Aβ  , the cell morphology changed, including the reduced cell quantity, membrane blebbing and cell shrinkage [22]. When pretreated with PPZM for 24 h, the morphology of the cells was observably improved, the above cell injuries alleviated.

PPZM Inhibited Aβ 25-25 -Induced Apoptosis in PC12 Cells
Flow cytometry and Hoechst/PI staining were further used to determine the effect of PPZM on Aβ 25-25 -induced apoptosis in PC12 cells. As shown in Figure 3, the percentage of apoptotic cells and the total apoptosis rate were significantly increased after treatment with Aβ 25-35 , pretreatment with PPZM (50, 100, and 200 µg/mL) effectively decreased the apoptosis rate (p < 0.01), indicating that PPZM might inhibit Aβ 25-35 -induced cell apoptosis in PC12 cells. As can be seen in Figure 4, the control cells showed even fluorescence with the regular shape and uniform size of nucleus. The nuclear morphology appeared as condensed bodies and highly fluorescent in the Aβ

PPZM Inhibited Aβ25-25-Induced Apoptosis in PC12 Cells
Flow cytometry and Hoechst/PI staining were further used to determine the effect of PPZM on Aβ25-25-induced apoptosis in PC12 cells. As shown in Figure 3, the percentage of apoptotic cells and the total apoptosis rate were significantly increased after treatment with Aβ25-35, pretreatment with PPZM (50, 100, and 200 μg/mL) effectively decreased the apoptosis rate (p < 0.01), indicating that PPZM might inhibit Aβ25-35-induced cell apoptosis in PC12 cells. As can be seen in Figure 4, the control cells showed even fluorescence with the regular shape and uniform size of nucleus. The nuclear morphology appeared as condensed bodies and highly fluorescent in the Aβ25-25 treated cells. Pretreatment with PPZM (50, 100, and 200 μg/mL) gradually relieved the apoptosis of the Aβ25-25 treated cells in a concentration-dependent manner.    Results are presented as the mean ± SD. ## p < 0.01 vs. the control group; ** p < 0.01 vs. the Aβ25-25 treated group.

PPZM Suppresses Aβ 25-35 -Induced Oxidative Stress
ROS levels were measured to evaluate the effects of PPZM against oxidative stress by flow cytometry by using DCFH-DA staining. As shown in Figure 5A, compared with the normal cells, the ROS level significantly increased in the Aβ 25-35 -treated cells (p < 0 01). However, pretreatment of PPZM (50, 100 and 200 µg/mL) markedly decreased the ROS level in Aβ 25-35 damaged PC12 cells (p < 0 05, p < 0 01, and p < 0 01). Effects of PPZM on the levels of SOD, LDH, and MDA in Aβ 25-35 -treated PC12 cells were further determined ( Figure 5B-D). The level of SOD was significantly decreased, whereas levels of LDH and MDA were obviously increased in Aβ 25-35 -treated PC12 cells (p < 0.01). PPZM treatment significantly increased the SOD level, and decreased the level of MDA and LDH at all the tested the concentrations with concentration-dependent manners.
the normal cells, the ROS level significantly increased in the Aβ25-35-treated cells (p < 0 01). However, pretreatment of PPZM (50, 100 and 200 μg/mL) markedly decreased the ROS level in Aβ25-35 damaged PC12 cells (p < 0 05, p < 0 01, and p < 0 01). Effects of PPZM on the levels of SOD, LDH, and MDA in Aβ25-35-treated PC12 cells were further determined (Figure 5B-D). The level of SOD was significantly decreased, whereas levels of LDH and MDA were obviously increased in Aβ25-35-treated PC12 cells (p < 0.01). PPZM treatment significantly increased the SOD level, and decreased the level of MDA and LDH at all the tested the concentrations with concentration-dependent manners.

PPZM Regulates the Expression of Apoptosis-Related and Nrf2/HO-1 mRNA
Effects of PPZM on expressions of apoptosis-related caspase-3, Bcl-2 and Bax mRNA in Aβ25-35-treated PC12 cells were shown in Figure 6. As shown in Figure 6A-C, compared with the normal cells, mRNA expressions of caspase-3 and Bax were significantly upregulated, whereas Bcl-2 was markedly downregulated in Aβ25-35-treated PC12 cells (p < 0.01). The changes in expression of mRNA level of Nrf2 were also analyzed via RT-qPCR. As shown in Figure 6D, different concentrations of PPZM significantly increased the mRNA expression of Nrf2 (p < 0.01) compared with the Aβ25-35 treated PC12 cells. The expression of the downstream factor HO-1 in this pathway was further investigated. As the results shown in Figure 6E, PPZM significantly upregulated the mRNA expression of HO-1 in Aβ25-35 treated cells at the concentrations of 50, 100, and 200 μg/mL (p < 0.01).

Polysaccharides Extraction
PZM were purchased from a local market in Hanyuan (ya'an, China). A specimen was stored at Shanxi Provincial Key Laboratory of Traditional Chinese Medicine Processing in Shanxi University of Chinese Medicine (Jinzhong, China). Polysaccharides from PZM were extracted by using the method in our previous report [21]. The obtained polysaccharides were redissolved and centrifuged (8000× g, 10 min), and then precipitated overnight by adding 95% ethanol (1:4, v/v). After being washed with anhydrous ethanol, acetone, and diethyl ether, the obtained polysaccharides (PPZM) were freeze-dried.

Enzymatic Digestion of PPZM
PPZM water solutions (5 mg/mL, 0.5 mL) were added to certain enzymes (the final concentration of pectinase and cellulase were 20 and 10 U/mL, respectively) and digested for 16 h at 37 • C. Then the solutions were heated at 80 • C for 10 min to stop the enzymatic digestion. After centrifugation (4000× g, 10 min), the supernatants were dried by using a nitrogen evaporator at 40 • C. The PPZM solution without enzyme digestion was served as blank control.

Derivatization with ANTS
The derivatization was performed by using the reported method with some modifications [23]. Briefly, ANTS was dissolved in acetic acid/water (3:17, v/v) to prepare a solution of 0.1 mol/L. NaCNBH 3 was prepared in DMSO (1 mol/L). Each dry enzymatic hydrolysate was added 125 µL of ANTS solution and 125 µL of NaCNBH 3 solution, respectively. The mixture was centrifuged and incubated at 37 • C for 17 h. Then, the solution was dried by using a nitrogen evaporator at 40 • C. The derivatized samples were resuspended in 0.5 mL of 25% glycerin solution and stored at −20 • C.

HPTLC Analysis
Sample separation (5 µL) was performed on 5 cm × 10 cm silica gel 60 plates (Merck, Darmstadt, Germany). Plates were first developed to a distance of 90 mm with ethyl acetate/glacial acetic acid/water (2:2:1, v/v/v) as mobile phase at room temperature. After being dried in air, the plates were redeveloped to a distance of 95 mm with the same mobile phase. Sugars were colorized with aniline-diphenylamine-phosphoric acid solution by heating at 105 • C for 10 min, and photographed.

PACE Analysis
PACE was performed according to the reported method [24]. In brief, all the samples (3-6 µL) were separated by using a mini-P4 vertical slab gel electrophoresis apparatus (Ji'nan Jun Yi Biotechnology Co., Ltd., Ji'nan, China). For separation of enzymatic hydrolysates, electrophoresis of 34% (w/v) polyacrylamide in the resolving gel with 8% (w/v) polyacrylamide in stacking gel was used. The 0.1 mol/L Tris-boric (pH 8.2) solution was applied as the electrophoresis buffer. The samples were electrophoresed at 15 mA to move the sample to the front end of the gel (observed with 365 nm UV lamp). Gels were imaged by using a 5000 Pro II gel imaging system (Guangzhou Biolight Biotechnology Co., Ltd., Guangzhou, China) under UV 365 nm.

Cell Culture
Rat adrenal pheochromocytoma PC12 cells were obtained from Procell Life Science &Technology Co., Ltd. (Wuhan, Hubei, China). Cells were cultured in DMEM supplemented with 10% FBS and 1% antibiotics (penicillin/streptomycin) in a humidified atmosphere of 5% CO 2 at 37 • C. The medium was changed every 2-3 days.

Treatment and Cell Viability
Cell viability assay was performed by using MTT assay [25]. PC12 cells (5 × 10 3 /well) were seeded into 96-well plates and cultured for 24 h. The medium was then replaced with PPZM at concentrations of 0, 50, 100, and 200 mg/mL. After being incubated for another 24 h, 10 µL MTT solution (the final concentration of 5 mg/mL) were added and incubated for 4 h. The culture medium was removed, and 150 mL DMSO was added. The absorbance at 490 nm was measured by a SpectraMax ® PLUS 384 microplate reader (Molecular Devices, Sunnyvale, CA, USA).

Flow Cytometry Analysis for Apoptosis and ROS
Cells were seeded into 6-well plates and incubated for 24 h. Then PPZM at final concentrations of 50, 100, and 200 µg/mL were added to the cells. After being cultured for 24 h, cells were treated with 20 µM Aβ 25-35 for another 24 h. Cells were harvested and washed by using PBS and stained by the Annexin V-APC/PI kit (Jiangsu KeyGEN BioTECH Co., Ltd., Nanjing, China). Cell apoptosis was detected by using a CytoFLEX flow cytometer (Beckman, Krefeld, Germany). In addition, the DCFH-DA ROS kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) was used to determine the intracellular ROS level by flow cytometry.

Cellular Apoptosis Analysis by Hoechst33342/PI Dual Staining
Cells (2 × 10 5 cells/well) were plated into 6-well plates for 24 h and then incubated with PPZM (50, 100, and 200 µg/mL) for an additional 24 h. Then, the cells were incubated with 20 µM Aβ 25-35 for 24 h. The Hoechst 33342 and PI fluorescent dye were added for 15 min, and the nuclear morphology was observed with a FV100 laser confocal microscope (Olympus, Tokyo, Japan).

Quantitative Real-Time Polymerase Chain Reaction (RT-qPCR) Assay
Total RNA of the PC12 cells was extracted by using TRIzol reagent. The purity and concentration of each RNA sample was determined by the OD value at 260 and 280 nm. RNA (2 µg) was reversely transcribed into cDNA by using the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher, Waltham, MA, USA). RT-qPCR analysis was performed by using an ABI StepOnePlus System (Applied Biosystems, Foster City, CA, USA). The mRNA data were normalized to GAPDH by using the 2 −∆∆CT method.

Statistical Analysis
Data obtained in the experiments are presented as mean ± standard deviations (SD). Statistical comparisons between groups were made by one-way analysis of variance (ANOVA) by using GraphPad Prism 9 software (GraphPad Software Inc., La Jolla, CA, USA). p < 0.05 was regarded as statistically different.

Discussion
As a neurodegenerative disease, AD brings an enormous financial burden for individuals and the society [26]. Therefore, it is urgent to make significant progress in order to find effective treatment for these diseases. Natural products isolated from plants have attracted much attention due to their high efficiency and biosafety, and polysaccharides are one of them. Many studies have shown that polysaccharides exhibit neuroprotective effects through a variety of mechanisms [27,28]. Experiments have shown that polysaccharides and polysaccharide-rich extracts can exhibit neuroprotective effects by promoting neurite outgrowth, and through Nrf2/HO-1, PI3K/Akt, NF-κB, and MAPK signaling pathways, etc. [29]. The present study indicated that PPZM alleviated apoptosis and oxidative stress induced by Aβ 25-25 through the inhibition of mitochondrial-dependent apoptosis and activation of Nrf2/HO-1 pathway. Although further experiments are needed in AD animal models to support the clinical application of PPZM, this study provides a new perspective for the therapeutic potential of PPZM in treating AD.
AD is the most common neurodegenerative disease in elderly people. Although great progress has been made in the mechanisms and treatment of AD, it is still an incurable disease [1]. Aβ directly or indirectly acts as a prooxidant, causing mitochondrial dysfunction and subsequent ROS generation [34]. Many studies reported that using antioxidants against Aβ-induced oxidative stress damage is a promising strategy to prevent AD [8]. In this study, the protective effects and possible mechanisms of PPZM in Aβ 25-35 -damaged PC12 cells were investigated. The results revealed that PPZM could inhibit Aβ 25-35 -induced cell damage by inhibiting apoptosis and reducing oxidative stress.
Apoptosis usually occurs in the process of aging. It exists as a stable defense mechanism to maintain the number of cells in the body [35]. Studies have shown that death receptor pathway and mitochondrial pathway are two main pathways related to apoptosis [36]. Previous research has also shown that Aβ [25][26][27][28][29][30][31][32][33][34][35] can cause cytotoxicity of PC12 cells by inducing apoptosis [37][38][39]. In the present study, Aβ 25-25 -induced apoptosis and the intervention of PPZM in PC12 cells were evaluated by flow cytometry and Hoechst/PI staining. It was found that treatment with PPZM significantly attenuated Aβ 25-35 -induced apoptosis in PC12 cells.
Mitochondria are the main site of ROS production, and oxidative stress is another important reason for Aβ 25-25 -induced cytotoxicity. The increase of ROS level in cells may lead to further pathological changes in brain neurons, which may lead to cognitive dysfunction. Studies have shown that in AD, oxidative stress can interfere with the process of mitosis, destroy cell cycles, and lead to apoptosis. In this study, we found that PPZM can play an antiapoptotic role in Aβ 25-25 -injured PC12 cells. We speculate that this effect may be related to the elimination of ROS by PPZM. The degree of oxidative damage can be measured by the LDH level, ROS scavenging enzyme SOD, and the final product of lipid peroxidation MDA [40,41]. Therefore, The LDH, SOD, and MDA levels were determined in this study to verify our hypothesis. As a result, PPZM significantly decreased the level of LDH and MDA, and increased the level of SOD in Aβ 25-25 -injured PC12 cells.
Mitochondria also play a key role in regulating the cell death pathway associated with Bcl-2 family protein members [42]. Bcl-2 is an antiapoptotic protein which inhibits apoptosis, while Bax is a proapoptotic protein that can induce apoptosis in neurons. Unbalanced Bax/Bcl-2 ratio can lead to increased mitochondrial membrane permeability and damaged mitochondrial integrity [43]. Cytochrome c is released from mitochondria to cytosol to activate caspase protein and lead to apoptosis [20]. In this study, PPZM treatment significantly increased the Bcl-2 mRNA expression, and decreased the mRNA expression of Bax; the Bax/Bcl-2 ratio was decreased in Aβ 25-25 -injured PC12 cells. In addition, the caspase-3 mRNA expression was also downregulated by PPZM. These results suggest that the protective effect of PPZM in Aβ 25-25 -injured PC12 cells may be related to the inhibition of mitochondrial-dependent apoptosis.
The Nrf2 pathway is a key pathway for cells to resist oxidation and maintain homeostasis [44]. Under normal physiological conditions, Nrf2 and Keap1 bind together to maintain a relative inhibition state. Under the condition of oxidative stress, Nrf2 released from Keap1 and entered the nucleus. Then Nrf2 combined with the antioxidant response element (ARE) to activate the expression of Nrf2 regulating genes and enhance the ability of cells to reduce oxidative stress [45,46]. Under the condition of oxidative stress, the lack or activation disorder of Nrf2 can increase intracellular ROS. Excessive active ROS will lead to oxidative damage of many molecules, including DNA, protein, unsaturated fatty acid, etc., leading to cell dysfunction, apoptosis, and even necrosis [47,48]. The antioxidant enzymes including nicotinamide adenine dinucleotide phosphate: quinine oxidoreductase-1 (NQO1), haemoxygenase-1 (HO-1), and SOD, etc. play an important role in protection of the cell from ROS damage [49,50]. In the present study, PPZM treatment markedly increased the mRNA expression of Nrf2 and HO-1. The results indicated that PPZM alleviated oxidative stress and apoptosis induced by Aβ  , which might be closely related to the Nrf2/HO-1 pathway.
This study demonstrated that PPZM protected PC12 cells against Aβ 25-35 -induced oxidative damage via inhibiting mitochondrial dependent apoptosis and activating Nrf2/HO-1 signal pathway. However, Western blot, antagonist or inhibitor, and molecular biological studies are needed to verify this pathway. In addition, more research can be done in the future for further application of PPZM in the treatment of AD. First, the role and mechanisms of PPZM in the treatment of AD need to be further verified through animal experiments by employing multiple models. Secondly, further separation and purification of PPZM should be done to obtain pure polysaccharides, and the structure of pure polysaccharides should be identified by a variety of technologies, such as mass spectrometry and nuclear magnetic resonance analysis. Lastly, the structure-activity relationship between the therapeutic effect of AD and the polysaccharides needs to be clarified.

Conclusions
In this study, PPZM were found to contain 1,4-α-D-galactosidic, 1,4-α-D-galactosiduronic, and (1→4)-β-D-glucosidic linkages. We demonstrated that PPZM significantly attenuated Aβ 25-35 -induced apoptosis in PC12 cells by decreasing the Bax/Bcl-2 ratio and downregulated caspase-3 expression. PPZM significantly decreased the level of LDH and MDA, and increased the level of SOD to suppress Aβ 25-35 -induced oxidative stress in PC12 cells. In addition, PPZM treatment markedly increased the mRNA expression of Nrf2 and HO-1 in Aβ 25-25 -injured PC12 cells. The results suggested that the protective effect of PPZM in Aβ 25-25 -injured PC12 cells may be related to the inhibition of mitochondrial-dependent apoptosis and alleviation of oxidative stress through the Nrf2/HO-1 pathway. PPZM can be used as a potential protective agent against Aβ 25-25 -induced neurotoxicity.